Hearing loss in humans and other mammals, whether congenital, as a result of loud noise exposure or due to ototoxic drugs, or age-related, is permanent. According to the World Health Organization (WHO), hearing loss affects 5% of the world's population (360 million people), while the current production of hearing aids only meet 10% of the demand. In the U.S., approximately 36 million adults report some degree of hearing loss, and two to three out of 1,000 babies born each year have detectable hearing loss according to the Center for Disease Control (CDC). In both cases, the social, emotional, and economic impacts affect the individuals' ability to communicate, resulting in reduced function in the workforce, isolation and depression, as well as increased financial costs.
The auditory sensory epithelium of the cochlea, also known as the organ of Corti, contains one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs), as well as surrounding supporting cells (SCs), including the several SC subtypes: Inner Border cells (IBs), Inner Phalangeal cells (IPhs), pillar cells (PCs) and Deiters' cells (DCs), which are directly underneath hair cells (HCs). During cochlear development, HCs and SCs are believed to share the same prosensory progenitors (Fekete, et al. (1998) J. Neurosci. 18:7811-21). Atonal protein homologue 1 (Atoh1) is a basic helix-loop-helix transcription factor required for HC development (Chen, et al. (2002) Development 129:2495-2505; Woods, et al. (2004) Nat. Neurosci. 7:1310-1318); no HCs appear in Atoh1 germline knock-out mice (Berminghan, et al. (1999) Science 284: 1837-1841).
When HC damage occurs in non-mammalian vertebrates, including birds and fish, their SCs spontaneously turn on Atoh1, and some change cell fate to become new HCs (Cafaro, et al. (2007) Dev. Dyn. 236:156-170); however, mammals have lost this capacity and suffer permanent deafness after HC damage (Brigande & Heller (2009) Nat. Neurosci. 12:679-685). In vitro ectopic expression of Atoh1 in the nonsensory greater epithelial ridge (GER) of neonatal rat cochlear explants generates ectopic HCs (Zheng & Gao (2000) Nat. Neurosci. 3:580-586), suggesting the sufficiency of Atoh1 in specifying a HC fate in a permissive cellular environment. In addition, in vivo overexpression of Atoh1 in mouse otocysts by electroporation (Gubbels, et al. (2008) Nature 455:537-541) and in the adult cochlea of guinea pigs by viral transduction (Izumikawa, et al. (2005) Nat. Med. 11:271-276) also leads to new HCs. However, this analysis did not indicate whether various postmitotic mouse SC subtypes, at different postnatal ages, are able to respond to ectopic Atoh1 expression and be converted into HCs. Recently, delivery of an γ-secretase inhibitor (LY411575) to noise-damaged adult mouse cochleae reportedly caused regeneration of functional hair cells and partial hearing recovery and this effect was reported to be linked to Atoh1 activation in the treated cochleae (Mizutari, et al. (2013) Neuron 77:58-69). However, it was not demonstrated whether these effects were specific to Atoh1 and supporting cells.
Ectopic expression of Atoh1 in mice has shown that PCs and DCs from neonatal and juvenile mice can be converted into HCs (Liu, et al. (2012) J. Neurosci. 32:6600-10). While these data demonstrate that neonatal and juvenile PCs and DCs can be converted into immature HCs, overexpression of Atoh1 alone was insufficient to complete the maturation of new HCs. Moreover, expression of Atoh1 in a spatially and temporally specific manner in the postnatal mammalian cochlea indicates that Atoh1 can generate cells in young animals with morphological, molecular, and physiological properties reminiscent of hair cells (Kelly, et al. (2012) J. Neurosci. 32:6699-6710). However, this competency is cell type specific and progressively restricted with age.
Screening assays for identifying Atoh1 activators and use of such activators for hair cell regeneration have been suggested (see U.S. Pat. No. 8,188,131). This patent describes small organic compounds that increase mouse Atoh1 expression and increase hair cell differentiation. Furthermore, U.S. Ser. No. 10/860,724 describes nucleic acids encoding Atoh1 for increasing Atoh1 expression and for the treatment of deafness, osteoarthritis, and abnormal cell proliferation.
It has also been reported that virus-mediated overexpression of Atoh1 and SKP2 (a presumed inhibitor of p27Kip1) induced ectopic HC formation in areas flanking the mature organ of Corti, although it was not demonstrated whether new HCs were generated from infected cells (Minoda, et al. (2007) Hearing Research 232:44-51). Similar inactivation of p27Kip1 in neonatal and juvenile SCs in mice resulted in their proliferation without transdifferentiation to HCs; such effect however failed in adult cochleae (Liu, et al. (2012) J. Neuroscience 32:10530-40; Oesterle, et al. (2011) Cell Cycle 10:1237-48).
p27Kip1 (also known as Cdkn1B) is a member of the Cip/Kip family of cell cycle inhibitors that are characterized by their ability to bind and inhibit cyclin dependent kinases (CDK)/cyclin complexes halting cell cycle progression in the G1 phase (Yoon, M. K., et al. (2012) Biochemical Society Transactions 40, 981-988). Because p27Kip1 inhibits the cell cycle, loss of p27Kip1 has been associated with some forms of cancer in humans and germline p27Kip1 deletion in mice results in sporadic pituitary tumors at old ages (Bertagnolli, M. M., et al. (2009) Clinical cancer research: an official journal of the American Association for Cancer Research 15, 2116-2122; Bottini, C., et al. (2009) International journal of oncology 34, 69-77; Chang, B. L., et al. (2004) Cancer research 64, 1997-1999; Slingerland, J., and Pagano, M. (2000) Journal of cellular physiology 183, 10-17; Pellegata, N. S., et al. (2006) Proceedings of the National Academy of Sciences of the United States of America 103, 15558-15563). Although, mutations in p27Kip1 may not always be causative of cancer, it is often dysregulated and associated with a poor prognosis (Chu, I. M., et al. (2008) Nature reviews. Cancer 8, 253-267; Rabbani, F., et al. (2007) BJU international 100, 259-263). Despite this, recent experiments have cast a light on how p27Kip1 may antagonize stem cells pluripotency (Menchon, C., et al., (2011) Cell Cycle 10, 1435-1447), and regenerative processes within certain tissue types giving some impetus to identification of small molecules to decrease the levels of p27Kip1. Specifically, loss of p27Kip1 has been associated with regenerative phenotypes in spinal cord injuries (Zhang, S., et al. (2013) J. Cellular Biochem. 114, 354-365), hepatocyte transplantation (Karnezis, A. N., et al. (2001) J of Clinical Investigation 108, 383-390), and in the inner ear (Liu, Z., et al. (2012) J. of Neuroscience: The Official J. of the Soc. for Neuroscience 32, 10530-10540; Oesterle, E. C., et al. (2011) Cell Cycle 10, 1237-1248; White, P. M., et al. (2011) Nature 441, 984-987; Mantela, J., et al. (2005) Development 132, 2377-2388). Interestingly, p27Kip1 initiates its expression during embryonic development coinciding with the exit of HCs and SCs within the organ of Corti from the cell cycle, and the beginning of quiescence during embryonic development (Chen, P. and Segil, N. (1999) Development 126, 1581-1590; Lee, Y. S., et al. (2006) Development 133, 2817-2826), implying a pivotal role for p27Kip1 in these post-mitotic cells. In vivo and explant studies in postnatal mouse cochleae revealed that removal of p27Kip1 from normally quiescent supporting cells forced these cells to re-enter the cell cycle (Liu, Z., et al. (2012) J. of Neuroscience: The Official J. of the Soc. for Neuroscience 32, 10530; Oesterle, E. C., et al. (2011) Cell Cycle 10, 1237-1248; Maas et al. (2013) JARO 14, 495), the first step towards replacing lost sensory cells. Similarly, multiple cell cycle inhibitors are up-regulated in older cells (Walters, B. J. and Zuo, J. (2013) Hearing Research 297, 68-83), implying that a cocktail of cell cycle inhibitors, including p27Kip1 may need to be developed to force proliferation to occur in older quiescent tissues.
Despite advances in research directed to inhibition of p27Kip1, there remains a significant need for compounds capable of inhibiting p27Kip1 expression for use in proliferation of cells, specifically hair cells, and methods of identifying and preparing same. These needs and other needs are satisfied by the present invention.